Parameters of subnuclear organization are critical for skeletal development and osteoblast differentiation. Our program initially identified Runx2 as the osteoblast-specific nuclear matrix protein NMP2. We developed the concept that the molecular function of Runx2 is to operate as a scaffolding protein that integrates regulatory signals from multiple osteogenic signaling pathways by modifying chromatin structure and supporting the organization and assembly of regulatory machinery for skeletal gene expression at strategic sites on target gene promoters and in focal nuclear microenvironments. We identified and structurally as well as functionally characterized a C- terminal intranuclear trafficking signal that directs Runx factors to nuclear domains where genes are activated or suppressed. Recently, we have established that Runx2 is bound to mitotic chromosomes to modulate both osteoblast specific gene expression and ribosomal RNA synthesis in osteoblasts immediately following mitosis. In collaborative studies with Projects 2 and 3, Project 1 will now pursue a highly integrated set of experimental approaches to examine the function of Runx2 as an epigenetic gene regulator that can control cell fate, lineage commitment and protein synthesis during both interphase and mitosis. We will determine the physiological consequences of abrogating the ability of Runx2 to integrate osteogenic signals at Runx2 subnuclear domains to control skeletogenesis in vivo, as well as osteoblast maturation and bone- specific gene expression ex vivo (Specific Aim 1). In addition, we will examine the osteoblast- related gene regulatory programs that depend on (i) subnuclear targeting of Runx2 during interphase and (ii)the epigenetic function of Runx2 that maintains a phenotypic regulatory memory during mitosis to control bone phenotypic genes as well as genes for cell cycle and growth control in progeny cells (Specific Aim 2). Furthermore, we will assess the bone specific molecular mechanisms by which Runx2 controls the anabolic activity of osteoblasts through regulation of osteoblast-specific genes (transcribed by RNA polymerase II) and ribosomal RNA genes (transcribed by RNA polymerase I) (Specific Aim 3). Relevance: Mechanisms that mediate the intranuclear organization of skeletal gene regulatory machinery provide necessary and novel options for targeted therapy.